The invention provides transgenic non-human animals and transgenic non-human mammalian cells harboring a transgene encoding an amyloid precursor protein (APP) comprising the Swedish mutation (lysine595-methionine596 mutated to asparagine595-leucine596); the invention also provides non-human animals and cells comprising a transgene encoding an APP comprising the Swedish mutation and further comprising functionally disrupted endogenous APP gene loci, transgenes and targeting constructs used to produce such transgenic cells and animals, transgenes encoding human Swedish mutation APP polypeptide sequences, and methods for using the transgenic animals in pharmaceutical screening and as commercial research animals for modeling neurodegenerative diseases (e.g., Alzheimer""s disease) and APP biochemistry in vivo.
Alzheimer""s disease (AD) is a progressive disease known generally as senile dementia. Broadly speaking the disease falls into two categories, namely late onset and early onset. Late onset, which occurs in old age (65+years), may be caused by the natural atrophy of the brain occurring at a faster rate and to a more severe degree than normal. Early onset AD is much more infrequent but shows a pathologically identical dementia with brain atrophy which develops well before the senile period, i.e., between the ages of 35 and 60 years.
Alzheimer""s disease is characterized by the presence of numerous amyloid plaques and neurofibrillary tangles (highly insoluble protein aggregates) present in the brains of AD patients, particularly in those regions involved with memory and cognition. While in the past there was significant scientific debate over whether the plaques and tangles are a cause or are merely the result of AD, recent discoveries indicate that amyloid plaque is a causative precursor or factor. In particular, it has been discovered that the production of xcex2-amyloid peptide, a major constituent of the amyloid plaque, can result from mutations in the gene encoding amyloid precursor protein, a protein which when normally processed will not produce the xcex2-amyloid peptide. It is presently believed that a normal (non-pathogenic) processing of the xcex2-amyloid precursor protein occurs via cleavage by a putative xe2x80x9cxcex1-secretasexe2x80x9d which cleaves between amino acids 16 and 17 of the protein. It is further believed that pathogenic processing occurs via a putative xe2x80x9cxcex2-secretasexe2x80x9d at the amino-terminus of the xcex2-amyloid peptide within the precursor protein. Moreover, xcex2-amyloid peptide appears to be toxic to brain neurons, and neuronal cell death is associated with the disease.
xcex2-amyloid peptide (also referred to as A4, xcex2AP, Axcex2, or Axcex2P; see, U.S. Pat. No. 4,666,829 and Glenner and Wong (1984) Biochem. Biophys. Res. Commun. 120: 1131) is derived from xcex2-amyloid precursor protein (xcex2APP), which is expressed in differently spliced forms of 695, 751, and 770 amino acids. See, Kang et al. (1987) Nature 325: 773; Ponte et al. (1988) Nature 331: 525; and Kitaguchi et al. (1988) Nature 331: 530. Normal processing of amyloid precursor protein involves proteolytic cleavage at a site between residues Lys16 and Leu17 (as numbered for the xcexdAP region where Asp597 is residue 1 in Kang et al. (1987)), supra, near the transmembrane domain, resulting in the constitutive secretion of an extracellular domain which retains the remaining portion of the xcex2-amyloia peptide sequence (Esch et al. (1990) Science 248:1122-1124). This pathway appears to be widely conserved among species and present in many cell types. See, Weidemann et al. (1989) Cell 57:115-126 and Oltersdorf et al. (1990) J. Biol. Chem. 265:4492-4497. This normal pathway cleaves within the region of the precursor protein which corresponds to the xcex2-amyloid peptide, thus apparently precluding its formation. Another constitutively secreted form of xcex2APP has been noted (Robakis et al. Soc. Neurosci. Oct. 26, 1993, Abstract No. 15.4, Anaheim, Calif.) which contains more of the xcex2AP sequence carboxy terminal to that form described by Esch et al. supra.
Golde et al. (1992) Science 255:728-730, prepared a series of deletion mutants of amyloid precursor protein and observed a single cleavage site within the xcex2-amyloid peptide region. Based on this observation, it was postulated that xcex2-amyloid peptide formation does not involve a secretory pathway. Estus et al. (1992) Science 255:726-728, teaches that the two largest carboxy terminal proteolytic fragments of amyloid precursor protein found in brain cells contain the entire xcex2-amyloid peptide region.
Recent reports show that soluble xcex2-amyloid peptide is produced by healthy cells into culture media (Haass et al. (1992) Nature 359:322-325) and in human and animal CSF (Seubert et al. (1992) Nature 359:325-327). Palmert et al. (1989) Biochm. Biophys. Res. Comm. 165:182-188, describes three possible cleavage mechanisms for xcex2APP and presents evidence that xcex2APP cleavage does not occur at methionine596 in the production of soluble derivatives of xcex2APP. U.S. Pat. No. 5,200,339, discusses the existence of certain proteolytic factor(s) which are putatively capable of cleaving xcex2APP at a site near the xcex2APP amino-terminus.
The APP gene is known to be located on human chromosome 21. A locus segregating with familial Alzheimer""s disease has been mapped to chromosome 21 (St. George Hyslop et al (1987) Science 235: 885) close to the APP gene. Recombinants between the APP gene and the AD locus have been previously reported (Schellenberg et al. (1988) Science 241: 1507; Schellenberg et al. (1991) Am. J. Hum. Genetics 48: 563; Schellenberg et al. (1991) Am. J. Hum. Genetics 49: 511, incorporated herein by reference).
The identification of mutations in the amyloid precursor protein gene which cause familial, early onset Alzheimer""s disease is evidence that amyloid metabolism is the central event in the pathogenic process underlying the disease. Four reported disease-causing mutations include with respect to the 770 isoform, valine717 to isoleucine (Goate et al. (1991) Nature 349: 704), valine717 to glycine (Chartier Harlan et al. (1991) Nature 353: 844), valine717 to phenylalanine (Murrell et al. (1991) Science 254: 97) and with respect to the 695 isoform, a double mutation changing lysine595-methionine596 to asparagine595-leucine596 (Mullan et al. (1992) Nature Genet 1: 345; Citron et al. (1992) Nature 360: 672) referred to as the Swedish mutation. APP alleles which are positively correlated with AD are termed xe2x80x9cdisease-associated allelesxe2x80x9d.
The development of experimental models of Alzheimer""s disease that can be used to define further the underlying biochemical events involved in AD pathogenesis would be highly desirable. Such models could presumably be employed, in one application, to screen for agents that alter the degenerative course of Alzheimer""s disease. For example, a model system of Alzheimer""s disease could be used to screen for environmental factors that induce or accelerate the pathogenesis of AD. In contradistinction, an experimental model could be used to screen for agents that inhibit, prevent, or reverse the progression of AD. Presumably, such models could be employed to develop pharmaceuticals that are effective in preventing, arresting, or reversing AD.
Unfortunately, only humans and aged non-human primates develop any of the pathological features of AD; the expense and difficulty of using primates and the length of time required for developing the AD pathology makes extensive research on such animals prohibitive. Rodents do not develop AD, even at an extreme age. It has been reported that the injection of amyloid protein (xcex2AP) or cytotoxic xcex2AP fragments into rodent brain results in cell loss and induces an antigenic marker for neurofibrillary tangle components (Kowall et al. (1991) Proc. Natl. Acad. Sci. (U.S.A.) 88: 7247). Mice which carry an extra copy of the APP gene as a result of partial trisomy of chromosome 16 die before birth (Coyle et al. (1988) Trends in Neurosci. 11: 390). Since the cloning of the APP gene, there have been several attempts to produce a mouse model for AD using transgenes that include all or part of the APP gene, unfortunately much of the work remains unpublished since the mice were nonviable or failed to show AD-like pathology. At least two published reports were retracted because of irregularities in reported results (Marx J Science 255: 1200; Wirak et al. (1991) Science 253: 323; Kawabata et al. (1991) Nature 354: 476; Kawabata et al. Nature 356: 23; Quon et al. (1991) Nature 352: 239; Marx Science 259: 457).
Thus, there is a need in the art for transgenic nonhuman animals harboring an intact disease-associated APP gene, either a human disease-associated allele such as a polynucleotide encoding a human APP protein comprising the Swedish mutation, or a complete genomic copy (or minigene) of the Swedish mutation APP gene.
Alternatively, a mutated rodent (e.g., murine) allele which comprises sequence modifications which correspond to a human APP sequence comprising the Swedish mutation can be substituted. Cell strains and cell lines (e.g., astroglial cells) derived from such transgenic animals would also find wide application in the art as experimental models for developing AD therapeutics and as a convenient source of APP protein comprising the Swedish mutation. Moreover, transgenic non-human animals comprising a transgene encoding a Swedish mutation APP protein and lacking functional endogenous APP gene loci (i.e., having an APP xe2x80x9cknockoutxe2x80x9d background) would be a convenient source of Swedish mutation APP protein in a background lacking other APP proteins that do not comprise the Swedish mutation.
Based on the foregoing, it is clear that a need exists for nonhuman cells and nonhuman animals harboring one or more transgenes encoding an APP gene comprising the Swedish mutation. Thus, it is an object of the invention herein to provide methods and compositions for transferring transgenes and homologous recombination constructs into mammalian cells, especially into embryonic stem cells. It is also an object of the invention to provide transgenic nonhuman cells and transgenic nonhuman animals harboring one or more Swedish mutation APP transgenes of the invention. Of further interest to the present invention are the application of such transgenic animals as in vivo systems for screening candidate drugs for the ability to inhibit or prevent the production of pathogenic xcex2-amyloid plaque. It would be desirable to provide methods and systems for screening test compounds for the ability to inhibit or prevent the conversion of amyloid precursor protein to pathogenic xcex2-amyloid peptide. In particular, it would be desirable to base such methods and systems on metabolic pathways which have been found to be involved in such conversion, where the test compound would be able to interrupt or interfere with the metabolic pathway which leads to conversion. Such methods and transgenic animals should provide rapid, economical, and suitable for screening large numbers of test compounds.
The references discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention.
In accordance with the foregoing objects, in one aspect of the invention are provided nonhuman animals harboring at least one copy of a transgene comprising a polynucleotide sequence which encodes a heterologous APP polypeptide comprising the Swedish mutation (asparagine595-leucine596) operably linked to a transcription regulatory sequence capable of producing expression of the heterologous APP polypeptide in the transgenic nonhuman animal. Said heterologous APP polypeptide comprising the Swedish mutation generally is expressed in cells which normally express the naturally-occurring endogenous APP gene (if present). Typically, the nonhuman animal is a mouse and the heterologous APP gene is a human Swedisch mutation APP gene. Such transgenes typically comprise a Swedish mutation APP expression cassette, wherein a linked promoter and, preferably, an enhancer drive expression of structural sequences encoding a heterologous APP polypeptide comprising the Swedish mutation.
The invention also provides transgenes comprising a gene encoding a Swedish mutation APP, said gene operably linked to a transcription regulatory sequence functional in the host transgenic animal (e.g., a neural-specific promoter). Such transgenes are typically integrated into a host chromosomal location by nonhomologous integration. The transgenes may further comprise a selectable marker, such as a neo or gpt gene operably linked to a constitutive promoter, such as a phosphoglycerate kinase (pgk) promoter or HSV tk gene. promoter linked to an enhancer (e.g., SV40 enhancer).
The invention further provides nonhuman transgenic animals, typically nonhuman mammals such as mice, which harbor at least one copy of a transgene or targeting construct of the invention, either homologously or nonhomologously integrated into an endogenous chromosomal location so as to encode a Swedish mutation APP polypeptide. Such transgenic animals are usually produced by introducing the transgene or targeting construct into a fertilized egg or embryonic stem (ES) cell, typically by microinjection, electroporation, lipofection, or biolistics. The transgenic animals express the Swedish mutation APP gene of the transgene (or homologously recombined targeting construct), typically in brain tissue. Such animals are suitable for use in a variety of disease model and drug screening uses, as well as other applications.
The invention also provides nonhuman animals and cells which harbor at least one integrated targeting construct that functionally disrupts an endogenous APP gene locus, typically by deleting or mutating a genetic element (e.g., exon sequence, splicing signal, promoter, enhancer) that is required for efficient functional expression of a complete gene product.
The invention also provides transgenic nonhuman animals, such as a non-primate mammal, that have at least one inactivated endogenous APP allele, and preferably are homozygous for inactivated APP alleles, and which are substantially incapable of directing the efficient expression of endogenous (i.e., wildtype) APP. For example, In a preferred embodiment, a transgenic mouse is homozygous for inactivated endogenous APP alleles and is substantially incapable of producing murine APP encoded by a endogenous (i.e., naturally-occurring) APP gene. Such a transgenic mouse, naving inactivated endogenous APP genes, is a preferred host recipient for a transgene encoding a heterologous APP polypeptide, preferably a human Swedish mutation APP polypeptide. For example, human APP comprising the Swedish mutation may be encoded and expressed from a heterologous transgene(s) in such transgenic mice. Such heterologous transgenes may be integrated in a nonhomologous location in a chromosome of the nonhuman animal, or may be integrated by homologous recombination or gene conversion into a nonhuman APP gene locus, thereby effecting simultaneous knockout of the endogenous APP gene (or segment thereof) and replacement with the human APP gene (or segment. thereof).